1. Technical Field
The subject invention relates to monovalent oligosaccharides and their use, for example, in the treatment and prevention of mammalian disease caused by Shiga toxin (ST) and Shiga-like toxin (S-LT). In particular, the trisaccharide globotriose (i.e., galactose xcex11,4 galactose xcex21,4 glucose) may be used to effectively prevent the pathological binding of ST and S-LT to target cells bearing the globotriaosylceramide glycolipid and to reverse this pathological binding once it has occurred.
2. Background Information
Shigellae are gram-negative, non-lactose-fermenting, non-motile bacilli of the family Enterobacteriaceae and are classified into four species. Shigella bacteria invade and kill intestinal epithelial cells causing colonic inflammation, muscosal ulceration and hemorrhage. Shiga Toxin (ST) is produced by Shigella dysenteria, the pathogen that causes dysentery. Secreted ST irreversibly inhibits protein synthesis by ADP-ribosylation of a key moiety of the ribosome. ST is cytotoxic for a variety of cell types, important among which are intestinal enterocytes, vascular endothelium and neurons.
Similar disease and cellular damage are caused by one distinct category of Escherichia coli which is present world-wide as a result of contaminated food or water. E. coli are gram-negative, lactose-fermenting motile bacilli of the family Enterobacteriaceae. Specifically, enterohemorrhagic E. coli (EHEC) produce a family of cytotoxins, termed Shiga-like toxins (S-LTs), which are closely related to those produced by Shigella. EHEC adhere to colonic enterocytes causing mucosal ulceration. Secretion of S-LT is essential to the pathogenesis of disease and results in hemorrhagic necrosis of affected tissue. As with ST, S-LTs can cross the intestinal mucosa to the circulation and bind to a variety of cells causing additional pathology. (Karen L. Kotloff, xe2x80x9cBacterial Diarrheal Pathogensxe2x80x9d. Advances in Pediatric Infectious Diseases, vol. 14 Mosby, Inc. 1999)
ST and S-LT are two members of a general class of bacterial AB5 toxins, which cause a spectrum of pathology in humans and animals. The AB5 toxins are characterized by a catalytic A subunit and a pentamer of B subunits that bind to specific glycolipids embedded in the exterior portion of the lipid bi-layer of animal cells. The binding of the B pentamer induces internalization of the holotoxin where the catalytic A subunit causes cytoxic damage. The animal and tissue tropism exhibited by the AB5 toxins is mediated by the binding specificity of the B subunit for particular oligosaccharide structures found on distinct glycolipids.
It is well accepted that the recognition motif for both ST and S-LT is the oligosaccharide portion of the globoseries-glycolipid globotriaosylceramide found on the exterior of a wide variety of mammalian cells. The free oligosaccharide (Galxcex11,4Galxcex11,4Glc) portion of globotriaosylceramide is known as globotriose. (Merritt E A and Hol W G J. AB5 toxins. Current opinion in structural biology 1995, 5:165-171).
The spectrum of clinical manifestations of gastrointestinal infection by bacteria that secrete ST and S-LT includes asymptomatic infection, nonbloody diarrhea, bloody diarrhea (hemorrhagic colitis), the hemolytic-uremia syndrome (HUS), thrombocytopenia purpura (TC), and death (Boyce T G, et al., 1995. The New England Journal of Medicine, 333 (6):364-368). ST and S-LT secreted in the intestinal lumen can be transported across the mucosa to circulation by transcytosis, where the toxin binds to and damages vascular endothelium and neuronal tissue. Circulating toxin results in a spectrum of pathologic events termed the hemolytic uremia syndrome (HUS). HUS is the most dreaded complication of toxin release and occurs in up to 10% of patients with this type of infection. It is most frequent in infants and the elderly, and is considered the most common cause of acute renal failure in children. Thrombocytopenia purpura is a similar condition typically diagnosed in adults, and is most likely HUS in infected individuals. HUS is characterized by microangiopathic hemolytic anemia, thrombocytopenia, renal-failure and central nervous system manifestations. The mortality rate for HUS is 3%-5%, and approximately 5% of surviving patients will have long-term renal and/or neurological damage.
Unfortunately, few antibiotics are clinically efficacious for Shigella infection, and resistance to first line antibiotic therapy (i.e., ampicilin) is increasing. Furthermore, antibiotic and anti-motility drugs are contraindicated for EHEC infections. Critically, there is no currently available treatment for HUS. Current management of HUS is restricted to supportive therapy and includes IV control of fluid and electrolyte balance, dialysis or transfusion. For these reasons new therapies are desperately needed. (Karen L. Kotloff, xe2x80x9cBacterial Diarrheal Pathogens. Advances in Pediatric Infectious Diseases, Vol. 14, Mosby, Inc. (1999)).
In principle, inhibiting the obligatory binding of the B pentamer of an AB5 toxin to its glycolipid ligand could prevent the toxin-induced tissue destruction associated with infection by AB5-toxigenic bacteria. However, the current belief in the field has been that this would be an ineffective approach for two reasons. First, monovalent oligosaccharides ligands have low binding affinity for proteins by which they are specifically bound. The standard view is well-characterized by the following statement: xe2x80x9cThe receptor-binding affinity of the individual B subunits for single ligands is relatively low; thus, a small molecule receptor analog cannot bind individual binding sites strongly enough to disable the toxinxe2x80x9d. (Donnelly et al., Nature Medicine, 2000. 6(3):257-258). Similarly, xe2x80x9cTherapies might target the obligatory oligosaccharide-toxin recognition event, but the low intrinsic affinity of carbohydrate-protein interactions hampers the development of low-molecular-weight inhibitorsxe2x80x9d (Kitov P I, et. al., Nature, 2000. 403:669-672). As a consequence of this widely accepted view, current technologies propose to use the oligosaccharides in a polyvalent presentation to more closely mimic the presentation found on a target cell (Armstrong et al. xe2x80x9cA Phase I Study of Chemically Synthesized Verotoxin (Shiga-like Toxin) Pk-trisaccharide Receptors Attached to Chromosorb for Preventing Hemolytic Uremia Syndromexe2x80x9d J. Inf. Dis. 171:1042-5 (1995), see also, U.S. Pat. No. 6,121,242).
A second difficulty associated with the use of a monovalent oligosaccharide ligand, specifically globotriose, has been cost-effective large scale synthesis. This problem has long been viewed as a major impediment to development of monovalent oligosaccharide based therapies (Walker W A, JPGN, March 2000).
In light of the above, new therapies are certainly required for the treatment of diseases and conditions caused by ST and S-LT. However, monovalent oligosaccharides have never been seriously considered as a possible treatment option until the present application.
All U.S. patents and publications referred to herein are hereby incorporated in their entirety by reference.
The present invention includes a composition comprising at least one monovalent oligosaccharide. The at least one monovalent oligosaccharide binds to Shiga-toxin and Shiga-like toxin. The composition may further comprise electolytes, and the at least one monovalent oligosaccharide may be, for example, globotriose, galabiose, globotetraose or the Forssamm antigen. The composition may be administered parenterally (e.g., intravenously) or enterally (e.g., orally) and may be a pharmaceutical or nutritional composition, such as a rehydration solution.
Additionally, the present invention also encompasses a method of treating or preventing a condition in a mammal, caused by Shiga toxin or Shiga-like toxin. The method comprises administering to the mammal, in need of the treatment or prevention, the composition described above of, in an amount sufficient to effect the treatment or prevention. The condition may be, for example, hemorrhagic diarrhea, hemolytic uremia syndrome or thrombocytopenia purpura. The Shiga toxin may be produced, for example, by Shigella dysenteria. The Shiga-like toxin may be produced, for example, by Escherichia coli. The mammal may be, for example, a human or a mouse or another entity fitting within the definition of a mammal.
Furthermore, the present invention also encompasses a method of preventing binding of a Shiga toxin or Shiga-like toxin to a cellular receptor for the Shiga toxin or Shiga-like toxin comprising the step of exposing the Shiga toxin or Shiga-like toxin and the cellular receptor to at least one monovalent oligosaccharide such that the Shiga toxin or Shiga-like toxin binds to the monovalent oligosaccharide instead of to the cellular receptor. The monovalent oligosaccharide may be as described above.
Also, the present invention includes a method of reversing the binding of Shiga toxin or Shiga-like toxin to a cellular receptor comprising the step of exposing the bound cellular receptor to at least one monovalent oligosaccharide for a time and under conditions sufficient to effect the reverse binding.
Again, the monovalent oligosaccharide used may be that described above.